Methods and devices for treating lung dysfunction

ABSTRACT

Methods and devices useful for treatment of lung conditions resulting from dysfunction of normal pulmonary physiology.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/291,210, filed May 15, 2001, and is a continuation of U.S. Ser. No.10/146,405, filed May 14, 2002 now U.S. Pat. No. 6,702,998, each ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods and devices for treating variousmedical conditions, e.g., lung dysfunction. Some of the conditions arerelated to cystic fibrosis, and others may be more widely applicable.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is the most common life-shortening genetic diseasein the white population. It occurs in the USA in about 1/3,300 whitebirths, 1/15,300 black births, and 1/32,000 Asian-American births; 30%of patients are adults. See, e.g., Berkow (ed.) The Merck Manual ofDiagnosis and Therapy Merck & Co., Rahway, N.J.; Thorn, et al.Harrison's Principles of Internal Medicine McGraw-Hill, N.Y.; andWeatherall, et al. (eds.) Oxford Textbook of Medicine Oxford UniversityPress, Oxford; the Cystic Fibrosis Foundation website (www.cff.org);Davis (ed. 1993) Cystic Fibrosis Marcel Dekker, ISBN: 082478815X; Dodge(ed. 1996) Cystic Fibrosis: Current Topics Wiley & Son, ISBN:0471963534; Bauernfeind, et al. (eds. 1996) Cystic Fibrosis PulmonaryInfections: Lessons from Around the World (Respiratory Pharmacology andPharmacotherapy) Springer Verlag, ISBN: 081765027X; Orenstein and Stern(eds. 1998) Treatment of the Hospitalized Cystic Fibrosis Patient MarcelDekker, ISBN: 0824795008; Hodson, et al. (eds. 2000) Cystic FibrosisOxford Univ. Press, ISBN: 0340742089; and Yankaskas and Knowles (eds.1999) Cystic Fibrosis in Adults Lippincott Pubs, ISBN: 0781710111. Seealso Conese and Assael (2001) “Bacterial infections and inflammation inthe lungs of cystic fibrosis patients” Ped. Infect. Dis. J. 20:207-213;Moss (2001) “New approaches to cystic fibrosis” Hosp. Pract. (Off. Ed.)36:25-27, 31-32, 35-37; Robinson (2001) “Cystic fibrosis” Thorax56:237-241; Ratjen (2001) “Changes in strategies for optimalantibacterial therapy in cystic fibrosis” Int. J. Antimicrob. Agents17:93-96; Hodson (2000) “Treatment of cystic fibrosis in the adult”Respiration 67:595-607; Doring, et al. (2000) “Antibiotic therapyagainst Pseudomonas aeruginosa in cystic fibrosis: a European consensus”Eur. Respir. J. 16:749-767; Beringer and Appleman (2000) “Unusualrespiratory bacterial flora in cystic fibrosis: microbiologic andclinical features” Curr. Opin. Pulm. Med. 6:545-550; Larson and Cohen(2000) “Cystic fibrosis revisited” Mol. Genet. Metab. 71:470-477; Nasr(2000) “Cystic fibrosis in adolescents and young adults” Adolesc. Med.11:589-603; Rahman and MacNee (2000) “Oxidative stress and regulation ofglutathione in lung inflammation” Eur. Respir. J. 16:534-554; and Kochand Hoiby (2000) “Diagnosis and treatment of cystic fibrosis”Respiration 67:239-247.

CF is typically carried as an autosomal recessive trait by about 3% ofthe white population. The most common gene responsible has beenlocalized to 250,000 base pairs of genomic DNA on chromosome 7q (thelong arm). This encodes a membrane-associated protein called the cysticfibrosis transmembrane regulator (CFTR). The most common gene mutation,F508, leads to absence of a phenylalanine residue at position 508 on theCFTR protein and is found on about 70% of CF alleles; >600 less commonmutations account for the remaining 30%. Although the exact function ofCFTR is unknown, it appears to be part of a cAMP-regulated Cl⁻ channeland appears to regulate Cl⁻ and Na⁺ transport across epithelialmembranes. Heterozygotes may show subtle abnormalities of epithelialtransport but are clinically unaffected.

Fifty percent of affected patients present with pulmonarymanifestations, usually chronic cough and wheezing associated withrecurrent or chronic pulmonary infections. Cough is typically the mosttroublesome complaint, often accompanied by sputum, gagging, vomiting,and disturbed sleep. Intercostal retractions, use of accessory musclesof respiration, a barrel-chest deformity, digital clubbing, and cyanosisoccur with disease progression. Upper respiratory tract involvementincludes nasal polyposis and chronic or recurrent sinusitis. Adolescentsmay have retarded growth, delayed onset of puberty, and a decliningtolerance for exercise. Pulmonary complications in adolescents andadults include pneumothorax, hemoptysis, and right heart failuresecondary to pulmonary hypertension.

Cystic fibrosis still presents major health problems to afflictedindividuals. It is a highly debilitating condition, and affectssignificant numbers of patients. As such, there is great need for newand more effective treatments. The present invention addresses these andother problems.

SUMMARY OF THE INVENTION

The present invention provides methods of treating a respiratory tractinfection in a primate suffering from cystic fibrosis or otherrespiratory tract condition, comprising administering to the primate aneffective amount of aerosolized thiocyanate or halides. In certainembodiments, the treating is after symptoms of bacterial infection havebeen detected or perhaps for viral infections instead or for bacterialinfections that follow viral infections of the respiratory tract.Typically, the lung infection is: Staphylococus aureus; Pseudomonasaeruginosa; or Burkholderia cepacia. The method may be used incombination, e.g., with a peroxidase; H₂O₂; and/or another treatment fora lung infection or cystic fibrosis. Such other treatments may be, e.g.,an antibiotic; an antiviral; an enzyme; or a manipulation, such asmassage or percussive therapy, including breathing exercises, posturaldrainage, chest percussion, vibration, or assisted coughing.

In another embodiment, the invention provides methods of treating arespiratory infection, e.g., lung, in a mammal, comprising administeringto the mammal an effective amount of thiocyanate or other halides, e.g.,I⁻, Br⁻, etc. Often, the administering is: by aerosol inhalation of thethiocyanate; or in combination with: an antibiotic; an antiviral; anenzyme; H₂O₂; or a medical manipulation. The lung infection is likely tobe: Staphylococcus aureus; Pseudomonas aeruginosa; or Burkholderiacepacia. In various embodiments, the effective amount of thiocyanate isbetween about 5 μM and 4 mM in the lung fluid; or the administeringoccurs between one administration in a week to hourly.

Another embodiment includes methods of treating a lung condition in aprimate or rodent suffering from or exhibiting symptoms of cysticfibrosis, the method comprising administering to the lung of thecreature an effective amount of thiocyanate. Typically, theadministering is by inhalation of aerosolized thiocyanate; the creatureis a mouse or rat; the creature suffers from a lung infection; or thetreating further includes administration of a peroxidase or H₂O₂.

Another embodiment includes methods of treating a lung condition in aprimate exhibiting symptoms of cystic fibrosis, comprising administeringto the respiratory system of the primate an effective amount of H₂O₂.Typically, the administering is by inhalation of H₂O₂, or a formulationwhich produces H₂O₂ in the respiratory system; or the lung conditioncomprises infection with a bacterium, e.g., Staphylococcus aureus,Pseudomonas aeruginosa, or Burkholderia cepacia, fungus, or virus.Often, the administering is in combination with administering aperoxidase, thiocyanate, or another treatment for a lung condition,e.g., a lung infection or cystic fibrosis, such as administering anantibiotic, anti-fungal, anti-viral, enzyme, e.g., a depolymerase, orapplying an airway clearance technique or physical therapy, e.g.,breathing exercises, postural drainage, chest percussion, vibration, orassisted coughing. The H₂O₂ is often administered at a concentrationbetween 10⁻⁷ M and 10⁻⁴ M in the lung fluid; or between once in a weekto hourly.

Another embodiment includes methods of treating a lung infection in amammal, the method comprising administering to the respiratory system ofthe mammal an effective amount of H₂O₂. Typically, the administering isby inhalation of H₂O₂; or a formulation is administered which producesH₂O₂ in the respiratory system; or the treatment prevents a minor orminimal lung infection from becoming more serious. Often the lunginfection comprises a bacterium, e.g., Staphylococcus aureus,Pseudomonas aeruginosa, or Burkholderia cepacia, fungus, or virus; orthe administering is in combination with peroxidase, thiocyanate, or anadditional treatment for a lung condition, including a lung infection orcystic fibrosis. The additional treatment will often include anantibiotic, anti-fungal, anti-viral, enzyme, e.g., a depolymerase, or anairway clearance technique or physical therapy, e.g., breathingexercises, postural drainage, chest percussion, vibration, or assistedcoughing. Typically the method will provide H₂O₂ at a concentrationbetween 10⁻⁷ M and 10⁻⁴ M in the lung fluid; or between once in a weekto hourly.

Yet another embodiment encompasses an inhaler comprising: hydrogenperoxide, a peroxidase, or thiocyanate. The inhaler may be labeled,e.g., include instructions, for administration to an individual withcystic fibrosis or other lung infection or condition. The inhaler mayfurther comprise an antibiotic, antifungal, or antiviral therapeutic.The individual may have a lung infection or other symptoms of cysticfibrosis.

DETAILED DESCRIPTION OF THE INVENTION Outline

-   -   I. Cystic Fibrosis    -   II. Current CF Treatments    -   III. The Lactoperoxidase (LPO) System    -   IV. Therapeutic Applications

In accordance with the objects outlined above, the present inventionprovides novel methods for treating or preventing signs or symptoms ofrespiratory dysfunction, particularly infections resulting fromcompromised airway functions related to resistance to infectious agentswhich enter the body through the airway mucosa. Devices are providedwhich are useful in addressing these problems.

Descriptions of lung immunity is applicable to other parts of therespiratory tract. Since the mechanisms described herein are common toairways in general, the applications described would also apply to otherairway mucosal surfaces.

I. Cystic Fibrosis

Cystic fibrosis is the most common genetic disorder among Caucasians.The disease is characterized by chronic respiratory infection thatbegins early in life with Staphylococcus aureus and Haemophilusinfluenzae infections and later colonization with mucoid strains ofPseudomonas aeruginosa. Chronic respiratory infection results inprogressive loss of lung function and greatly diminished quality of lifeor shortened life expectancy. See references listed in the Backgroundsection. Chronic infection implies failure of airway host defenseagainst bacterial colonization although the exact defects in hostdefense remain unclear. The normal airway barrier to infection iscomplex and multifactorial. The currently accepted view is that ciliamechanically clear potential infectious particles and that mucusprovides both a protective barrier and serves as a carrier with theproper viscoelastic properties to permit ciliary movement. Theprotective barrier of mucus is thought to be both physical and chemicalas a number of anti-microbial components are secreted by airwayepithelial and submucosal gland cells. Lung physiology and function arefundamental clinical study areas, and are described, e.g., in Murray andNadel (2001) Textbook of Respiratory Medicine Saunders, Philadelphia,Pa. (ISBN: 0721692532); Crystal, et al. (1996) The Lung LippincottWilliams & Wilkins, Philadelphia, Pa. (ISBN 0-397-51632-0); Brewis, etal. (1995) Respiratory Medicine 2d Ed., Saunders, Philadelphia, Pa.(ISBN: 0702016411); and Fraser, et al. (1988) Diagnosis of Diseases ofthe Chest Volume 3d Ed., Saunders, Philadelphia, Pa. (ISBN: 0721638708).

Additionally, nearly all exocrine glands are affected in varyingdistribution and degree of severity. Involved glands are of 3 maintypes: those that become obstructed by viscid or solid eosinophilicmaterial in the lumen (e.g., pancreas, intestinal glands, intrahepaticbile ducts, gallbladder, submaxillary glands); those that arehistologically abnormal and produce an excess of secretions (e.g.,tracheobronchial and Brunner's glands); and those that arehistologically normal but secrete excessive Na⁺ and Cl⁻ (e.g., sweat,parotid, and small salivary glands). Duodenal secretions are viscid andcontain an abnormal mucopolysaccharide. The reproductive functions forboth adult males and females are typically also affected.

Morbidity in cystic fibrosis (CF) primarily results from chronicrespiratory infections by Pseudomonas aeruginosa. Mutations in CFTRresult in an anion channel defect but how this leads to impaired hostdefense against infection is not fully understood. Evidence suggeststhat the lungs are histologically normal at birth. Pulmonary damage isprobably initiated by diffuse obstruction in the small airways byabnormally thick mucus secretions. Bronchiolitis and mucopurulentplugging of the airways occur secondary to obstruction and infection.Bronchial changes are more common than parenchymal changes. Emphysema isnot prominent. As the pulmonary process progresses, bronchial wallsthicken; the airways fill with purulent, viscid secretions; areas ofatelectasis develop; and hilar lymph nodes enlarge. Chronic hypoxemiaresults in muscular hypertrophy of the pulmonary arteries, pulmonaryhypertension, and right ventricular hypertrophy. Much of the pulmonarydamage may be caused by immune-mediated inflammation secondary to therelease of proteases by neutrophils in the airways. Bronchoalveolarlavage fluid, even early in life, contains large numbers of neutrophilsand increased concentrations of free neutrophil elastase, DNA, andinterleukin-8.

Early in the course, Staphylococcus aureus is the pathogen most oftenisolated from the respiratory tract, but as the disease progresses,Pseudomonas aeruginosa is most frequently isolated. A mucoid variant ofPseudomonas is uniquely associated with CF. Colonization withBurkholderia cepacia occurs in up to 7% of adult patients and may beassociated with rapid pulmonary deterioration.

II. Current CF Treatments

Many of the signs and symptoms of CF are pulmonary. Thus, chest x-rayfindings often aid diagnosis. Hyperinflation and bronchial wallthickening are typically the earliest findings. Subsequent changesinclude areas of infiltrate, atelectasis, and hilar adenopathy. Withadvanced disease, segmental or lobar atelectasis, cyst formation,bronchiectasis, and pulmonary artery and right ventricular enlargementoccur. Branching, fingerlike opacifications that represent mucoidimpaction of dilated bronchi are characteristic. In almost all cases,sinus x-rays and CT studies show persistent opacification of theparanasal sinuses.

Pulmonary function tests typically reveal hypoxemia and reduction inforced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1),and FEV1/FVC ratio and an increase in residual volume and the ratio ofresidual volume to total lung capacity. Fifty percent of patients haveevidence of airway hyperreactivity.

Current treatment for pulmonary manifestations includes prevention ofairway obstruction and prophylaxis against and control of pulmonaryinfection. Prophylaxis against pulmonary infections consists ofmaintenance of pertussis, and immunity to Haemophilus influenzae,varicella, measles, and influenza by vaccination. In unvaccinatedpatients, amantadine can be used for prophylaxis against influenza A.

Chest physical therapy consisting of postural drainage, percussion,vibration, and assisted coughing is recommended at the first indicationof pulmonary involvement. In older patients, alternative airwayclearance techniques such as active cycle of breathing, autogenicdrainage, flutter valve device, positive expiratory pressure mask, andmechanical vest therapy may be effective. For reversible airwayobstruction, bronchodilators may be given orally and/or by aerosol andcorticosteroids by aerosol. O₂ therapy is indicated for patients withsevere pulmonary insufficiency and hypoxemia. Noninvasive positivepressure ventilation by nasal or facemask also can be beneficial.Long-term daily aerosol administration of dornase alfa (recombinanthuman deoxyribonuclease) has been shown to slow the rate of decline inpulmonary function and to decrease the frequency of severe respiratorytract exacerbations. Other aspects of current treatment are known. See,e.g., The Merck Manual, and CF references listed above.

Drug therapies include oral corticosteroids for infants with prolongedbronchiolitis and in those patients with refractory bronchospasm,allergic bronchopulmonary aspergillosis, and inflammatory complications(e.g., arthritis and vasculitis). Long-term use of alternate-daycorticosteroid therapy can slow the decline in pulmonary function, butbecause of steroid-related complications it is not recommended forroutine use. Patients receiving corticosteroids must be closelymonitored for evidence of carbohydrate abnormalities and linear growthretardation. Ibuprofen, when given at a dose sufficient to achieve apeak plasma concentration between 50 and 100 μg/ml over several years,has been shown to slow the rate of decline in pulmonary function,especially in children 5 to 13 yr. The appropriate dose must beindividualized based on pharmacokinetic studies.

Antibiotics should be used in symptomatic patients to treat bacterialpathogens in the respiratory tract, according to culture and sensitivitytesting. A penicillinase-resistant penicillin (e.g., cloxacillin ordicloxacillin) or a cephalosporin (e.g., cephalexin) is the drug ofchoice for staphylococci. Erythromycin, amoxicillin-clavulanate,ampicillin, tetracycline, trimethoprim-sulfamethoxazole, or occasionallychloramphenicol may be used individually or in combination forprotracted ambulatory therapy of pulmonary infection due to a variety oforganisms. Ciprofloxacin is effective against sensitive strains ofPseudomonas. For severe pulmonary exacerbations, especially in patientscolonized with Pseudomonas, parenteral antibiotic therapy is advised,often requiring hospital admission but safely conducted at home incarefully selected patients. Combinations of an aminoglycoside(tobramycin, gentamicin) with an anti-Pseudomonas penicillin are givenIV. Intravenous administration of cephalosporins and monobactams withanti-Pseudomonas activity also may be useful. Serum aminoglycosideconcentrations should be monitored and dosage adjusted to achieve a peaklevel of 8 to 10 μg/ml (11 to 17 μmol/L) and a trough value of <2 μg/ml(<4 μmol/L). The usual starting dose of tobramycin or gentamicin is 7.5to 10 mg/kg/day in 3 divided doses, but high doses (10 to 12 mg/kg/day)may be required to achieve acceptable serum concentrations. Because ofenhanced renal clearance, large doses of some penicillins may berequired to achieve adequate serum levels. The goal of treatingpulmonary infections should be to improve the clinical statussufficiently so that continuous use of antibiotics is unnecessary.However, in some ambulatory patients with frequent pulmonaryexacerbations, long-term use of antibiotics may be indicated. Inselected patients, long-term tobramycin therapy by aerosol may beeffective.

Aerosol therapy with ribavirin should be considered in infants withrespiratory syncytial viral infection. Levels of airway glutathione havebeen shown to be reduced in the airway lavages and in plasma of cysticfibrosis patients. In vitro studies of airway lining cells from normaland cystic fibrosis patients have shown that cystic fibrosis cells aredefective in glutathione transport compared to normal and thatreplacement of the defective gene product restores glutathionetransport. Since the LPO system can oxidize glutathione and sincemicroorganisms have specific uptake systems for glutathione, and sinceglutathione is deficient in cystic fibrosis, combination therapies withall or part of the LPO system and glutathione may provide increasedefficacy or may be necessary for system function.

That lung infections are problematic in CF is evidenced by the recentactivity in this area. The first new drug therapy developed exclusivelyfor CF in 30 years was approved by the Food and Drug Administration(FDA) in 1993. In clinical trials, this mucus-thinning drug calledPulmozyme®, reduced the number of respiratory infections and improvedlung function. In 1995, a four-year CF Foundation-supported study showedthat the drug, ibuprofen, reduced the rate of lung inflammation inchildren with CF—under controlled conditions, and in high doses. This isa DNAse that liquifies secretions that are containing large amounts ofbacterial DNA.

In late 1997, the FDA approved the drug TOBI™ (tobramycin solution forinhalation). In clinical trials, this reformulated version of the commonantibiotic improved lung function in people with CF and reduced thenumber of hospital stays. The benefits of TOBI™ are that it can bedelivered in a more concentrated dose directly to the site of CF lunginfections more efficiently, and that it is preservative-free. Thedevelopment of TOBI™ should lead to a long line of other aerosolizedantibiotics for people with CF.

Beyond currently available antibiotics, the CF Foundation pursues novelstrategies that will lead to entirely new forms of antibiotics, i.e., toclear lung infections. The promising compound, IB367, represents one ofan up-and-coming new class of drugs that should provide physiciansunique tools to better manage chronic CF lung infections.

In CF cells, salt does not move properly because the protein product ofthe CF gene is defective—and makes a faulty channel for the salt(chloride) to exit. Scientists are therefore looking for ways to get thechloride out of cells. INS365 is being evaluated for its ability tostimulate cells to secrete chloride. This, in turn, should lead to mucusthat is less thick and sticky.

Other therapeutics in development are described in the Cystic FibrosisFoundation website. Among Phase III clinical trial candidates areprotein-assist and chloride channel strategy therapeutics andanti-inflammatory and anti-infection therapeutics.

The protein-assist and chloride channel strategies attempt to correctthe defective CFTR protein or at least get enough protein to the cellsurface, where it can operate as a channel. For people who do not haveCF, the CFTR protein acts as a “one-way door” that allows cells torelease chloride. However, for people with CF, this protein isdefective, which leads to improper chloride balance and the thick,sticky CF mucus. Chloride channel therapies are another exciting methodof correcting the defect in CF cells. Researchers believe that gettingCF cells to release chloride would be of a tremendous therapeuticbenefit for CF patients.

Anti-inflammatory and anti-infection therapies are directed tomedications that help control infection and inflammation because theseCF symptoms tear down airways in a dangerous cycle. When CF lungs becomeinfected, they also become over-inflamed. This over-inflammation leadsto a greater susceptibility to future infection. Breaking or preventingthis destructive cycle is a key focus for CF research. Although thesetherapies do not target the basic defect in CF cells, there are manyexciting medications being evaluated that could extend lives by stemmingthe course of dangerous infections and the resulting accumulation ofdamage.

The present invention provides another therapeutic. While not limitedaccording to mechanism, the present invention would seem to be based, inpart, upon insight into the natural systems of lung immunity whichappear to be compromised in CF.

III. The Lactoperoxidase (LPO) System

The lactoperoxidase (LPO) antibiotic system, shown to participate inovine airway host defense, requires the anion thiocyanate (SCN⁻) inairway secretions for its function. To test the hypothesis that CFmutations lower airway [SCN⁻] thereby leading to impaired LPO-mediatedhost defense, human airways were examined for an LPO system. Normal andCF epithelial cell cultures were compared for differences in SCN⁻transport that could be functionally linked to CF. LPO and SCN⁻ werefound in human airway secretions and LPO mRNA was found in tracheatissue. In air liquid interface cultures, normal human airway epitheliatransported SCN⁻ from the basolateral to the apical compartment andconcentrated the anion at the mucosal surface with pharmacologicalproperties consistent with CFTR-mediated channel activity. Thepharmacological characteristics of SCN⁻ transport implied at least anindirect role of CFTR. SCN⁻ transport and apical SCN⁻ accumulation weresignificantly reduced in CF cultures. This alteration in SCN⁻ transportsuggests a simple mechanistic link between CF mutations and the LPO hostdefense system in human airway.

The cystic fibrosis gene product, cystic fibrosis transmembraneregulator (CFTR), contains an anion channel that has been extensivelycharacterized and mutations result in loss of functional CFTR anionchannel activity on the apical cell surface. The current controversyregarding the link between cystic fibrosis and defective airway hostdefense has been reviewed by others and centers on the exactdetermination of the ionic composition of airway surface liquid (ASL)and the volume of ASL in cystic fibrosis versus normal airways. Opposingviews focus on either the effects of dehydration and impaired ciliaryclearance of mucus or the effects of hypertonic ASL on the activity ofantimicrobials in mucus. Both views are limited by our incompleteknowledge regarding the total array of mechanisms at work in airway hostdefense.

For example, the lactoperoxidase (LPO) system has recently been shown tobe important to clear bacteria from sheep airways (Gerson, et al. (2000)Am. J. Respir. Cell Mol. Biol. 22:665-671) although its existence inhuman airways has not previously been shown. The LPO antibiotic systemworks to preserve milk sterility and contributes antibiotic activity tosaliva. LPO uses H₂O₂ to catalyze oxidation of the anions SCN⁻ (apseudohalide) and some halides (e.g., I⁻ and Br⁻) to the antibioticforms (e.g., OSCN⁻, OI⁻, or OBr⁻). Thomas, et al. (1991), pp. 123-142,in Everse, et al (eds.) Peroxidases in Chemistry and Biology.Descriptions will be directed to SCN⁻, but similarly, I⁻ and Br⁻ may besubstituted.

Since it is known that the LPO system is effective against pseudomonads(Bjorck, et al. (1975) Applied Microbiology 30:199-204), and that SCN⁻is a requisite substrate for LPO and is permeant through CFTR(Tabcharani, et al. (1993) Nature 366:79-82), defects in SCN⁻ transportcould be, at least partially, responsible for the chronic respiratorybacterial infections seen in CF patients. Described experiments hereinsupport the hypothesis that the LPO system is present in human airwaysand that cystic fibrosis airway epithelia are defective in SCN⁻transport to the airway surface.

The experiments presented here document the presence of thelactoperoxidase anti-microbial system in human airways that was notpreviously recognized as a factor in human airway host defense. The dataalso show that the secretion of LPO's substrate, SCN⁻, appears to rely,at least indirectly, on the CFTR anion channel and that mutant CFTRresults in defective delivery of LPO's substrate to the airway lumen.

Pseudomonas infection occurs with high frequency in CF airways and theLPO system's efficacy against pseudomonads has been previouslydocumented. Bjorck, et al. (1975) Applied Microbiology 30:199-204.Defective SCN⁻ transport in CF may impair the correct functioning of theLPO antibiotic system and be a contributor to the characteristic chronicrespiratory infections seen in this disease. SCN⁻ is elevated in CFsweat (Gibbs and Hutchings (1961) Proc. Soc. Exp. Biol. Med.106:368-369) and others have suggested that SCN⁻ might be a pathogenicfactor in CF based on decreased serum levels in severely affected CFpatients compared to normal individuals (which would predict an evenlower concentration in CF airways than that calculated above). Weuffen,et al. (1991) Padiatrie und Grenzgebiete 30:205-210.

Several mouse models of cystic fibrosis have been developed and noneshow the characteristic chronic airway infection seen in the humandisease. Murine respiratory tracts were examined for the presence of LPOor an LPO-like protein by collecting tracheal secretions and assayingenzyme activity. Attempts to demonstrate an LPO system in airways ofnormal mice failed, e.g., in mice whose airways were challenged withlive bacteria, or in mice previously sensitized and challenged withovalbumin in order to up-regulate secretory cells in the airways. SinceLPO is made by airway submucosal glands and goblet cells (Gerson, et al.(2000) Am. J. Respir. Cell Mol. Biol. 22:665-671; and Salathe, et al.(1997) Am. J. Respir. Cell Mol. Biol. 17:97-105), the lack of thesestructures in mouse airways suggested that LPO may not be present inthis animal's respiratory tract and that mice may rely on an alternateairway defense. Lack of reliance on an LPO system could explain, inpart, why CFTR knockout mice do not display the chronic respiratoryinfection characteristic of cystic fibrosis.

One unexplained feature of chronic infection in CF is the inability ofneutrophils to clear bacteria. CF airways are characterized by aconstant high level of lumenal neutrophils that appear to be competentin phagocytosis, chemotaxis, and have functional myeloperoxidase (MPO).Yet bacterial infection is not resolved even in the presence ofaggressive antibiotic therapies, suggesting a defect in neutrophilkilling. Although mucoid strains of Pseudomonas are present in laterstages of CF, defects in host defense and neutrophil mediated killingexist in earlier stages of the disease suggesting that mucoid characteralone does not explain the inability of neutrophils to resolveinfections. MPO uses Br⁻, I⁻, Cl⁻, and SCN⁻ as substrates and althoughmost studies of MPO-mediated bacterial killing have concentrated on useof Cl⁻, SCN⁻ has recently been shown to be the preferred substrate ofMPO. Thus impaired SCN⁻ transport to the lumen could potentially resultin defects in both the epithelial-derived LPO system as well as theneutrophil-derived MPO antibacterial activity. The SCN⁻ deficiency maybe doubly detrimental to maintenance of a sterile airway.

Because the LPO antimicrobial system relies on correct SCN⁻ anionconcentration rather than osmolarity of ASL or water volume, measures ofthese ASL characteristics are not expected to be relevant to LPOantibacterial activity. The total loss of airway lumenal SCN⁻ wouldprobably not be detected in measurement of ASL ionic strength ortonicity as it is ≦1 mM in ASL of normal airways. Elemental analysis ofASL has demonstrated large amounts of sulfur that have been ascribed tothe presence of sulfated mucins and that may obscure the detection ofSCN⁻ in these studies.

Taken together with the extensive literature on mutant CFTR-linkedchanges in the airway, the data presented here suggest that defectiveSCN⁻ transport to the airway may account for a portion of the chronicinfection seen in cystic fibrosis and that aerosol SCN⁻ therapy maybolster airway host defense against infection in patients with thisdisease.

Since the LPO system has been shown to be functional in the ovineairways and in secretions collected from human airways (Wijkstrom-Frei,et al. (2003) Am. J. Respir. Cell and Mol. Biol. 29:206-212), not onlySCN⁻, but also H₂O₂ concentration in the airway surface liquid isimportant in full expression of LPO system activity.

IV. Therapeutic Application

According to the present invention, methods for treating variousrespiratory system conditions are provided, particularly those relatedto respiratory system response to infectious agents. These conditionsare typical of the normal or dysfunctional respiratory tract in amammal, e.g., primate, sheep, rodent, or common domestic pet, includingdogs and cats. The present disclosure provides evidence for a naturalresponse to airway infections in primates. This system, the LPO system,is implicated as a mechanism to decrease likelihood of establishedcolonization of microbes in the lung.

These reagents can be combined for therapeutic use with additionalactive or inert ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, e.g., immunogenic adjuvants, along withphysiologically innocuous stabilizers, excipients, or preservatives.These combinations can be sterile filtered and placed into dosage formsas by lyophilization in dosage vials or storage in stabilized aqueouspreparations.

The LPO system is found in the lung of at least some species. In thosespecies, including certain primates, the presence of that system shouldprovide antibacterial function, which should be generally effectiveagainst most common infectious bacterial and fungal species. Theenzymatic conversion of the anion SCN— to the antimicrobial OSCN—resultsin a means to decrease microbial load to the lung. Other species, whichlack the LPO system, may serve as species useful for testing whether theintroduction of such a system may be of value in certain lung infectionmodels.

Since the lung fluid, which bathes the mucosa, is the target oftreatment, an aerosol is the preferred method of administration. Whileother means to administer to the lung will be available, there is agreat deal of inhaler art. Here, with the administration of smallmolecules, or perhaps in combination with a biologic, the inhalers arepreferred means for administration. See, e.g., U.S. Pat. No. 6,223,746“Metered dose inhaler pump”; U.S. Pat. No. 6,205,999 “Methods andapparatus for storing chemical compounds in a portable inhaler”; U.S.Pat. No. 6,204,054 “Transcytosis vehicles and enhancers for drugdelivery”; U.S. Pat. No. 6,202,643 “Collapsible, disposable MDI spacerand method”; U.S. Pat. No. 6,196,219 “Liquid droplet spray device for aninhaler suitable for respiratory therapies”; U.S. Pat. No. 6,196,218“Piezo inhaler”; U.S. Pat. No. 6,182,655 “Inhaler for multiple dosedadministration of a pharmacological dry powder”; U.S. Pat. No. 6,180,663“Therapeutic nasal inhalant”; U.S. Pat. No. 6,176,238 “Dispenser forsubstances in powder or granular form”; U.S. Pat. No. 6,170,482“Inhalation apparatus”; U.S. Pat. No. 6,165,484 “EDTA and otherchelators with or without antifungal antimicrobial agents for theprevention and treatment of fungal infections”; U.S. Pat. No. 6,164,275“Inhaler carrier”; U.S. Pat. No. 6,158,428 “Infant inhaler”; U.S. Pat.No. 6,155,251 “Breath coordinated inhaler”; U.S. Pat. No. 6,153,224“Carrier particles for use in dry powder inhalers”; U.S. Pat. No.6,153,173 “Propellant mixture for aerosol formulation”; U.S. Pat. No.6,149,892 “Metered dose inhaler for beclomethasone dipropionate”; U.S.Pat. No. 6,142,146 “Inhalation device”; U.S. Pat. No. 6,142,145“Inhalation device”; U.S. Pat. No. 6,140,323 “Dosing method ofadministering medicaments via inhalation administration or skinadministration”; and U.S. Pat. No. 6,138,673 “Inhalation device andmethod”. Many others may be found by a simple search through a patentdatabase.

As such, the treatment described herein is intended to decrease themicrobial load in an infected lung by a significant and measurableamount or to reduce the frequency of infection establishment in a normallung exposed to infectious agents. While the normal lung may possesssufficient LPO to convert inhaled thiocyanate to an activatedantimicrobial product, certain lung conditions may deplete thethiocyanate, H₂O₂ or LPO, whose supplementation may overcome dysfunctionresulting therefrom. Thus, the treatment is intended to decrease themicrobial flora in an infected airway by at least about 10%, preferablyat least about 20%, more preferably by at least about 50%, and better byat least 2, 3, 5, or 7 fold. Other relevant endpoints would be sputumneutrophil counts, sputum volume production, pulmonary function testing,incidence of hospitalization among a population of patients, or chestX-ray evaluation, which would improve by similar measures, e.g. 10%,20%, 50%, or multifold, according to treatment. Such measures will betaken at appropriate time points, as clinically significant. Such may beat specified time points after microbial exposure or introduction, andmay range from minutes, hours, days, or weeks after defined identifiableevents.

Treatment dosages should be initially titrated in development tooptimize safety and efficacy. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof these reagents. Animal testing of effective doses for treatment ofparticular disorders will provide further predictive indication of humandosage. Various considerations are described, e.g., in Gilman, et al.(eds.) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,latest Ed., Pergamon Press; and Remington's Pharmaceutical Sciences,latest ed., Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for direct application to thelung. Pharmaceutically acceptable carriers will include water, saline,buffers, and other compounds described, e.g., in the Merck Index, Merck& Co., Rahway, N.J. Dosage ranges for SCN⁻ would ordinarily be expectedto deliver amounts that result in mucosal surface liquid concentrationslower than 10 mM concentrations, typically less than about 4 mMconcentrations, preferably about 400 μM, preferably not less than about4 μM. Slow release formulations, or a slow release apparatus may beutilized for continuous or long term administration. See, e.g., Langer(1990) Science 249:1527-1533. Dose evaluation may depend, e.g., onpatient weight, age, condition, and other relevant variables. Exemplarydose ranges should account for total ASL and estimated sputum volumes.It is expected that normal amounts would be about 400 μM. The balance ofexcess activity versus tolerable damage from excess may depend onpatient condition. Final concentrations would likely be in the 40 μM to400 μM range.

Dosage amount for H₂O₂ would ordinarily be expected to deliver amountsthat result in mucosal surface liquid concentrations lower than 100 μMconcentrations, e.g. in concentrations which may be in the range of 0.1to 1 or 10 μM.

Therapeutic formulations may be administered in many conventional dosageformulations. While it is possible for the active ingredient to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. Formulations typically comprise at least one activeingredient, as defined above, together with one or more acceptablecarriers thereof. Each carrier should be both pharmaceutically andphysiologically acceptable in the sense of being compatible with theother ingredients and not injurious to the patient. Formulations includethose suitable for lung administration. The formulations mayconveniently be presented in unit dosage form and may be prepared bymany methods well known in the art of pharmacy. See, e.g., Gilman, etal. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral MedicationsDekker, N.Y.; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms:Tablets Dekker, N.Y.; and Lieberman, et al. (eds. 1990) PharmaceuticalDosage Forms: Disperse Systems Dekker, N.Y. The therapy of thisinvention may be combined with or used in association with other agents,e.g., other lactoperoxidase substrates or therapies.

As indicated above, various combinations of therapeutics may be used,e.g., with antibiotics, antifungal agents, antiviral agents, withvarious peroxidases, with sulfhydryl compounds, or other therapeutics orprocedures used in the treatment of CF or other lung infections. Suchprocedures include airway clearance techniques (ACTs) and physiotherapy,e.g., breathing exercises, postural drainage, chest percussion,vibration, assisted coughing, and forced expiratory techniques. Otherlung conditions will include those where infections may be problematic,e.g., tuberculosis, asthmatic conditions, artificially ventilatedpatients, HIV or other immunosuppressed individuals. As described above,there are various treatments for cystic fibrosis, which may be combinedwith the treatment described herein. Administration may be simultaneousor sequential, e.g., with the various other therapeutics, or with theDNAse (Pulmozyme®) or TOBI™.

Certain precautions would be indicated when administering thiocyanateand/or H₂O₂. To limit the possibility of thiocyanate toxicity, othersources of thiocyanate should be carefully monitored. Dietary sourcesinclude consumption of cauliflower, Brussels sprouts, and othercyanogenic compounds, e.g., various seeds from squashes such as pumpkin.Smoking, exposure to welding fumes, or diesel exhaust should becontrolled. The monitoring of blood thiocyanate levels may be useful,including determination of swallowed and absorbed compound. Serum andurine thiocyanate should be measured at appropriate times, e.g., 1, 4, 8h following initial administration, and perhaps during treatment.Clinical parameters may also be monitored, e.g., body temperature,volume or rate of respiration, various subjective measures of lunginfection or cystic fibrosis symptoms.

Side effects of treatment may include expected fever from bacteriallysis. Excessive levels of SCN⁻ may itself lead to toxic effects on thehost.

Indications for use of the invention will include other respiratorytract conditions, e.g., beyond CF. Because of the ease of administrationand low cost of the materials in the treatment described, thesupplementation of the endogenous barrier to lung infections may beuseful in many contexts. The circumstances in which lung infections maybe problematic are well known in the art. See, e.g., Crystal, et al.(1996) The Lung Lippincott, ISBN 0-397-51632-0; and other lungreferences.

Thus, the present invention may become adopted as standard means totreat generic respiratory tract infections as a first and immediatestep.

EXAMPLES Example I General Methods

Collection of Secretions and Tissues

Airway secretions were obtained from intubated patients undergoingambulatory outpatient elective surgery according to IRB approvedprotocols. Patients were selected for not having respiratory disease.Saline (3 ml) was injected into the tracheal tube and immediatelysuctioned into a trap. Recovered secretions were spun at 16000 rpm for20 min. at 4° C. The supernatant was aliquoted and stored at −80° C. forlater analysis.

Western Blotting

Anti-LPO antiserum was prepared by immunizing rabbits withchromatographically purified sheep airway LPO (Gerson, et al. (2000) Am.J. Respir. Cell Mol. Biol. 22:665-671) that was further enriched byexcision and elution from SDS gels. Affinity purification of LPOspecific antibodies was performed using bovine milk LPO (Sigma Chemical)coupled to Sepharose. Control antibodies were IgGs prepared fromun-immunized rabbits.

PCR

Human tracheal mRNA was purchased from Clontech (Palo Alto, Calif.).ds-cDNA was made using the SuperScript kit from Life Technologies(Bethesda, Md.) and size fractionated. Transformation into E. coli DH10αgave 5×10⁵ independent colonies. Degenerate oligonucleotides weredesigned from conserved regions of mammalian heme peroxidases.

Cell Culture and Thiocyanate Transport Experiments

Airway epithelial cell cultures were grown and differentiated on either6.5 mm or 24 mm collagen-coated T-clear membranes (Costar #3450) at anair-liquid interface. Bernacki, et al. (1999) Am. J. Respir. Cell Mol.Biol. 20:595-604. Experiments comparing non-CF and CF cells wereperformed with cultures grown simultaneously and matched in passagenumber, the number of cells plated, and days in culture. Culturestypically had a resistivity≧300 Ω·cm. Apical surfaces of cultures werewashed with PBS and then incubated overnight with 70 μM ¹⁴C—SCN⁻ (50μCi/mM) in the basolateral media. To initiate experiments, apicalsurfaces of the cultures were rapidly washed three times with Dulbecco'sPBS (50 μl for 6.5 mm filters or 500 μl for 24 mm filters) containing100 μM amiloride. Following a third wash, additional aliquots wereplaced on the apical surface for sequential 2 min incubations at 37° C.in humidified 5% CO₂. PKA stimulation of SCN⁻ efflux was demonstrated byadding 500 μM dibutryl cAMP and 10 μM forskolin to the PBS washes. Thestimulated efflux was inhibited by further addition of 500 μMglibenclamide to washes containing dibutryl cAMP and forskolin.Basolateral media were sampled after the last wash and ¹⁴C—SCN⁻ in mediaand apical washes was determined by liquid scintillation counting. Thecollected ¹⁴C—SCN⁻ was soluble following 10% TCA precipitation showingthat radiolabel was not covalently attached to protein.

Example II LPO System in CF Patients

A hypothesis that the LPO system may be defective in cystic fibrosis wastested. Human airways were tested for the presence of an LPO antibioticsystem. To assay for the presence of LPO in the airway lumen withnegligible contamination by saliva (that also contains LPO), secretionswere collected by suctioning intubated patients who were undergoingelective surgery and did not have active pulmonary disease. Assays ofthese secretions (Thomas, et al. (1994) J. Dental Res. 73:544-555; andSalathe, et al. (1997) Am. J. Respir. Cell Mol. Biol. 17:97-105) showedthe presence of LPO activity and Western blot analysis of trachealsecretions demonstrated the presence of anti-LPO immuno-reactive bands,equivalent in MW_(app) to that reported for human milk (Shin, et al.(2000) J. Nutr. Biochem. 11:94-102) and salivary (Mansson-Rahemtulla, etal. (1988) Biochemistry 27:233-239) LPO.

To demonstrate that the LPO detected by enzymatic assays was made in theairway, human tracheal mRNA was used to construct a cDNA library. Twodegenerate oligonucleotides complementary to conserved regions ofmammalian heme peroxidases were used to screen the library by PCR. Thisidentified a partial cDNA (GenBank:AF027971) identical to human salivary(see GenBank:U39573) and milk LPO (see GenBank:M58151). These datademonstrated that human airways synthesized and secreted enzymaticallyactive LPO into the airway lumen.

To function as an antibiotic system, LPO requires both SCN⁻ and H₂O₂ assubstrates. H₂O₂ has been identified in human airways previously. SeeWang, et al. (1996) Proc. Nat'l Acad. Sci. U.S.A 93:13182-13187;Dohlman, et al. (1993) Am. Rev. Respir. Dis. 148:955-960; Antczak, etal. (1997) Eur. Respir. J. 10:1235-1241; and Jobsis, et al. (1997) Eur.Respir. J. 10:519-521. SCN⁻ is found in salivary and gastric secretionsbut has not been conclusively demonstrated in human airways. Previously,reports of SCN⁻ in human sputum (Dacre and Tabershaw (1970) Arch. ofEnviron. Health 21:47-49) were believed to represent salivacontamination because SCN⁻ was below detection levels in airwaysecretions collected by bronchoscopic lavage. Since the large dilutionsassociated with bronchoalveolar lavage also resulted in false negative[SCN⁻] in sheep, SCN⁻ was measured in human tracheo-bronchial secretionscollected without dilution from patients with no apparent lung diseaseintubated for reasons other than respiratory failure in the medicalintensive care unit. SCN⁻ assays (Densen, et al. (1967) Arch. Environ.Health 14:865-874) revealed the presence of detectable SCN⁻. Theconcentration was within the ranges reported in saliva and gastricsecretions, ruling out that the SCN⁻ measured was due to minor amountsof saliva contamination. The measured [SCN⁻] was high enough to serve asa substrate for LPO (Pruitt, et al. (1988) Biochemistry 27:240-245) andthus, together with the presence of LPO and H₂O₂, comprises an intactLPO antibiotic system in human airways.

Serum SCN⁻ is thought to be derived primarily from diet either directlyor by conversion from cyanide. Since mean normal non-smoking plasma SCN⁻values (40-50 μM; see Lundquist, et al. (1995) Eur. J. Clin. Chem. Clin.Biochem. 33:343-349) are substantially lower than the levels measured inairway secretions, the data suggest that SCN⁻ is actively accumulated inairway secretions.

There are several possible approaches to determine if CF airwaysecretions might contain less SCN⁻. Direct analysis of these fluids iscomplicated by high viscosity and infection. High viscosity makesaccurate sample collection difficult and this may explain theconflicting reports (Boucher (1994) Am. J. Respir. Crit. Care. Med.150:271-281) from previous analyses of ionic composition of CFsecretions. Infection results in high concentrations ofneutrophil-derived myeloperoxidase that also oxidizes SCN⁻ (Thomas andFishman (1986) J. Biol. Chem. 261:9694-9702). For these reasons,epithelial cell cultures were chosen for these experiments to testwhether CF airway secretions might be deficient in their ability (orlack thereof) to transport SCN⁻ from the serosal (basolateral) to themucosal (apical) surface.

Example III In vitro Evaluation of LPO System Relating to CF

Bronchial epithelial cells were obtained from either CF or non-CF lungsat the time of transplant or organ donation. Cells were passaged twice(thereby de-differentiated) and then cultured at an air-liquid interfacefor re-differentiation. See Bernacki, et al. (1999) Am. J. Respir. CellMol. Biol. 20:595-604. Between 1 and 3 weeks after exposure of theapical surface to air, the cultures were secreting mucus and cilia werepresent. To measure the flux of SCN⁻ across the epithelium, ¹⁴C—SCN⁻ (70μM) was added to the basolateral media and 18-24 h later, its rate ofefflux onto the apical surface liquid was monitored. Following threerapid PBS washes of the apical surface, additional PBS aliquots wereplaced on the apical surface for sequential 2 min incubations. Theappearance of ¹⁴C—SCN⁻ in washes was used to measure efflux rates.Airway epithelial cell cultures from three separate CF patients werecompared to three non-CF control cultures. In each case, CF cells showedsignificantly reduced (˜4 fold) SCN⁻ efflux rates.

Since CFTR mutants have alterations in anion channel activity, compoundsknown to stimulate or inhibit CFTR activity were used to evaluate apossible contribution of CFTR to the SCN⁻ efflux in cultures. Non-CFcultures showed an increase in efflux after stimulation of PKA bydibutryl cAMP (0.5 mM) and forskolin (10 μM), while CF cultures did not.In non-CF cells, the PKA-stimulated and baseline SCN⁻ efflux was blockedby glibenclamide (500 μM), but not by4,4′-diisothiocyanostilbene-2,2′disulphonic acid (DIDS) or4,4′-diinitrostilbene-2,2′disulphonic (DNDS). The glibenclamideinhibition was reversible. In CF cells, on the other hand, neitherglibenclamide nor DIDS had any effect. Thus SCN⁻ efflux in the culturesresembled the anion channel activity of CFTR with regard to response tothese agents.

The differences in SCN⁻ efflux between non-CF and CF cultures wereindependent of the age of the cultures (repeated measurements from 2-8week old cultures), suggesting that a possible difference in time toreach full differentiation between CF and non-CF cells was notresponsible for these observations. The fact that efflux was stimulatedby cAMP and reversibly sensitive to glibenclamide argues strongly thatthe measured transepithelial SCN⁻ flux was not due to leakage via aparacellular route. The same properties, along with the insensitivity todisulphonic stilbenes, suggest that, at the very least, the measuredSCN⁻ efflux is indirectly related to functional CFTR and perhaps evencarried by CFTR itself. This notion is further supported by the factthat CF epithelial cells did not carry SCN⁻ efficiently.

Exact determination of accumulated apical [SCN⁻] was not possible inthese experiments because the naturally present small apical surfacevolumes in the air-liquid interface cultures normally prevented accuratesampling or even measurements by dilution. However comparison of thetotal amounts of SCN⁻, recovered in the initial PBS wash of the apicalsurface after overnight incubations with ¹⁴C—SCN⁻ in the basolateralmedia, showed 11-fold higher levels in non-CF compared to CF cultures.Although CF cultures are reported to have about one half the amount ofapical surface fluid when compared to non-CF (Matsui, et al. (1998) Cell95:1005-1015), the difference in accumulated apical ¹⁴C—SCN⁻ between CFand non-CF cultures would remain significant (˜5.5 fold) if this volumedifference is taken into account. Thus, it appears that non-CFepithelial layers accumulate SCN⁻ to significantly higher levels thanCF. Interestingly, on the occasions that apical surface liquid wasvisible on non-CF cultures that had been pre-incubated with basolateral¹⁴C—SCN⁻, direct sampling (2-5 μl) allowed measurement of the [SCN⁻] andshowed that it was concentrated ˜6 fold over the 0.07 mM in thebasolateral media to 0.4 mM. This concentration of SCN⁻, measured inundiluted non-CF apical surface liquid, is similar to levels measured inundiluted airway secretions collected from patients. Liquid was neverobserved on the apical surface of CF cultures, as expected. Matsui, etal. (1998) Cell 95:1005-1015. Therefore, direct measurement of [SCN⁻] inCF apical surface fluid could not be made. However, by estimating thevalues for CF cells using the difference to non-CF cells measured above(˜5.5 fold), the apical SCN⁻ concentration appears likely to be close tothat in the basolateral media.

Since these results suggested that SCN⁻ was concentrated in the apicalsurface liquid over that in the basolateral media in normal cellcultures, the effect of increasing [SCN⁻] in the apical PBS washes wastested on SCN⁻ efflux in these cells. Non-CF cultures transported SCN⁻into apical PBS washes containing 1-5 mM SCN⁻, showing that the cellswere able to concentrate ¹⁴C—SCN⁻.

The measured/estimated difference in apical [SCN⁻] between CF and non-CFairway epithelial cells is expected to be functionally significant sinceprevious studies on bovine milk LPO kinetic properties (Pruitt, et al.(1988) Biochemistry 27:240-245) show that a 5 fold decrease in the[SCN⁻] in this concentration range could potentially result in, a 100fold decrease in LPO enzyme activity. Thus, the disparity in SCN⁻transport and apical concentration between CF and non-CF epithelia,reported here, would be expected to have a dramatic effect on theefficacy of this host defense mechanism. This implication could not betested using collected apical surface liquid because these cultures didnot synthesize and secrete reproducibly measurable LPO.

Example IV LPO System Serves Antibacterial Function

Human airway secretions were obtained from intubated patients undergoingambulatory outpatient elective surgery according to IRB approvedprotocols. Patients were selected for not having respiratory disease.Saline (3 ml) was injected into the tracheal tube and immediatelysuctioned into a trap. Recovered secretions were spun at 16,000 rpm for20 min. at 4° C. The supernatant was aliquoted and stored at −80° C. forlater analysis. Undiluted secretions were obtained from acutely (<24 h)intubated patients in the medical ICU who were intubated for reasonsother than respiratory disease. Selected patients had no clinical signsof respiratory infection. Secretions were cleared by centrifugation(100,000×g, 30 min), aliquoted, and stored at −80° C.

Assay of Antibacterial Activity

Pseudomonas aeruginosa (ATCC 27853) were grown in LB broth overnight at37° C. in a rotary shaker. Bacteria were collected in the stationaryphase of growth and further diluted in LB broth to 5-6×10⁴/ml. Afteradding glycerol to 15%, aliquots of bacteria were stored at −80° C.

Cultures of non-CF and CF airway epithelial cells were matched withregard to passage number, days in culture, and days at the air liquidinterface. At least 24 h prior to the experiments, basolateral media ofsome cultures were supplemented with 100 μM KSCN. The apical surfaces ofthe cultures were washed with 0.25 ml PBS and the washes were pooled andstored at −20° C. SCN⁻ dependent antibiotic activity was assayed using560 μl of apical culture washes, adjusted to pH 5.7, and containing2400-3600 P. aeruginosa. Since LPO was not secreted by the under theseculture conditions, assays of washes also contained 1.125 μg/ml LPO and10⁻⁵ M H₂O₂. Control experiments were performed using only PBS adjustedto pH 5.7. Growth of bacteria was not affected by addition of 1.125μg/ml LPO, 10⁻⁵ M H₂O₂, or 5×10⁻⁴ M SCN⁻ singly or in pairs. However,addition of all three reconstituted a functional LPO system that wasbactericidal. To show thiocyanate dependence, in some experiments CFculture secretions were also supplemented to 5×10⁻⁴ M SCN⁻. Mixtureswere sampled immediately and 4 h after incubation at room temperatureand CFU were determined by plating on LB Agar. Antibacterial activitywas expressed as a ratio of CFU after 4 h to the starting CFU in thesample to control for slight differences in the starting number ofbacteria.

LPO-dependent antibiotic activity of airway secretions obtained duringsurgery was determined using similar mixtures that were supplementedonly with exogenous 10⁻⁵ M H₂O₂ and not LPO or thiocyanate. LPOdependence of antibacterial activity in human airway secretions wasdemonstrated by its requirement for added H₂O₂ and sensitivity to 10⁻³ Mdapsone in separate incubations.

To demonstrate that the LPO system has antibacterial functions in humanairways, secretions were tested for their ability to prevent growth ofbacteria in vitro. P. aeruginosa, that were diluted into PBS, pH 5.7,and incubated for 4 h at room temperature, increased in cell number by afactor of 2.1±0.3 (mean±SE) (n=6). Given the measured concentrations ofLPO and SCN⁻ in these secretions, existing H₂O₂ is expected to beconsumed shortly after collection and thus no H₂O₂ was detectable (<10⁻⁷M) in the samples. It is unlikely that H₂O₂ would continue to beproduced in secretions after collection since its source is thought tobe cellular in origin. For this reason, secretions were supplementedwith 10⁻⁵ M H₂O₂. Addition of H₂O₂ to 10⁻⁵ M or dapsone to 10⁻³ M had nomeasurable effect on cell growth (1.9±0.3, n=6). However when bacteriawere diluted into airway secretions of six different patients in thepresence of 10⁻⁵ M H₂O₂, cell growth was completely inhibited(0.98±0.16, n=6). Inclusion of dapsone, a potent inhibitor of LPO,significantly blocked the H₂O₂-dependent bacteriostatic properties ofthe airway secretions (1.68±0.31, n=6, p<0.05 compared to no dapsone).The H₂O₂ dependence and dapsone sensitivity of the bacteriostaticactivity supports the conclusion that the LPO system functions in humanairway as an antibacterial defense.

Example V Thiocyanate Transport Defects and In Vitro AntibacterialActivity

To assess the effects of reduced SCN⁻ transport on LPO-mediatedantibacterial activity, the apical surfaces of cultures were washed withPBS and then the washes were used in LPO antibacterial assays. The dayprior to washing, SCN⁻ (100 μM) was added to the basolateral media toallow transport to the apical surface. Since culture conditions did notresult in LPO synthesis and secretion, washes were supplemented withLPO. H₂O₂ was added to 10⁻⁵ M, a concentration that did not alone orwith LPO, give rise to any antibacterial activity in washes from cellscultured in the absence of basolateral SCN⁻. However, incubation of P.aeruginosa with LPO/H₂O₂ supplemented washes from non-CF cultures grownwith basolateral SCN⁻ resulted in LPO-dependent killing of bacteria. Incontrast, incubation of P. aeruginosa under the same conditions withwashes from CF cultures had no detectable LPO-dependent effect. Additionof exogenous SCN⁻ (0.5 mM) to culture washes generated antibacterialactivity in CF samples, demonstrating that the lack of antibacterialactivity was the result of defective SCN⁻ transport by the CF epithelialcells. Addition of the same amount of SCN⁻ to the non-CF culture washesincreased the LPO dependent activity in response to increased [SCN⁻].Thus, the lack of SCN⁻ transport by CF cells compromised the in vitroLPO system reconstituted in apical washes of the ALI cultures.

Example VI Airway LPO System Requires Continuous H₂O₂

To demonstrate that the airway LPO system requires continuous H₂O₂production to function, LPO dependent killing was measured at pH 5.7,6.2, and 6.8 using bovine milk LPO as a model for the airway LPO.Concentrations of the LPO system components were selected to be withinthe measured ranges in human airways. Bovine milk LPO (6.5 mg/ml), SCN⁻(0.4 mM) and H₂O₂ (10⁻⁵ M) at all pH's tested effectively blocked thegrowth of P. aeruginosa and Burkholderia cepacia that frequentlycolonize cystic fibrosis airways. At pH 5.7 and 6.2, the activity wasbactericidal, while at pH 6.8, it was bacteriostatic. Additionalcontrols showed that the antibacterial activity was dependent on thepresence of SCN⁻ and H₂O₂. It was sensitive to heating the LPO to 100°C. prior to use and was inhibited by the peroxidase inhibitors dapsone(10⁻³ M) or salicyl hydroxamic acid (10⁻⁴ M). All of thesecharacteristics were consistent with LPO-mediated antibacterialactivity.

Since the pK of the LPO product HOSCN/OSCN⁻ is about 5.3, the loss ofbactericidal activity at higher pH could be due to the lowerconcentration of HOSCN. To increase the antibacterial product, theenzymatic consumption of H₂O₂ during experiments was compensated for atpH 6.8 by measuring the consumption of H₂O₂ at 0.5 h or 1 h intervalsand replenishing to restore it to 10⁻⁵ M. Under these conditions the LPOsystem was bactericidal at pH 6.8 suggesting that replenishing H₂O₂increased LPO activity required for bactericidal activity. Thus, it isexpected that a constant supply of H₂O₂ in the airway for LPO's useresults in an effective antibacterial system against P. aeruginosa andB. cepacia.

To demonstrate that human airway epithelia produce H₂O₂, enzymaticmachinery was identified in these cells that produces H₂O₂ and H₂O₂production by human airway epithelia re-differentiated in culture at anair-liquid interface was measured. Reverse transcription-polymerasechain reaction (RT-PCR) was performed with primers designed to detectany mammalian NADPH oxidase capable of producing H₂O₂ through superoxideintermediate. This RT-PCR yielded a cDNA fragment that when sequencedwas 99% identical with human Dual Oxidase 1 (Duox1), the large molecularweight NADPH oxidase homologue. To further confirm these results, genespecific primers chosen within the 5′ UTR and 3′ UTR of homo sapiensDual Oxidase 1 (Genbank accession No 017434) were used for RT-PCR andgenerated the complete 5.5 Kb cDNA sequence having 100% identity withhuman Duox1.

As expected from identification of Duox1 in the cells, H₂O₂ was producedat the apical surface of airway epithelial cells as detected using aquantitative fluorometric-micro-assay based on horseradish peroxidasecatalyzed H₂O₂ oxidation of N-acetyl-3,7 dihydroxyphenoxazine. Previousstudies on porcine thyroid plasma membrane preparations that containedDuox1 demonstrated that NADPH oxidase activity was calcium-dependent inthese membranes. See Nakamura, et al. (1987) J. Biochem. (Tokyo)102(5):1121-32). Since the predicted molecular structure of Duox1 and 2identified an intracellular loop containing 2 EF-hand calcium-bindingmotifs, the Ca²⁺ stimulated H₂O₂ production was assessed. ALI cultureswere stimulated for one hour with Thapsigargin (1 μM), an inhibitor ofthe endoplasmic reticulum calcium ATPase pump that increasesintracellular Ca²⁺. Concomitant with the increment of the intracellularcalcium concentration, the apical H₂O₂ produced by stimulated cells alsoincreased from a baseline value of 59±16 H₂O₂ pmoles/hr to 128±17 H₂O₂pmoles/hr, for control and thapsigargin, respectively. To confirm thatthis H₂O₂ production was due to a NADPH oxidase-like activity, the cellswere treated with thapsigargin in the presence of 1 μM ofDiphenyleneiodonium (DPI), a well known NADPH oxidase inhibitor thatdisrupts the activity of the flavoprotein oxidoreductase domain. In thisassay, the cells were incubated for one hour with either DPI alone or incombination with thapsigargin (1 μM). These results demonstrated thatthe inhibitory effect of DPI blocked the thapsigargin induced increasein apical H₂O₂ production (45±19 H₂O₂ pmoles/hr). The demonstration ofDPI inhibitable apical H₂O₂ generation under the control of cytosoliccalcium concentration is consistent with the presence of Duox1 in airwayepithelia. The data support the idea that stimulation of Duox1 producesH₂O₂ for the LPO mediated antibiotic activity.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. An inhaler comprising a mouthpiece and containing a composition andadapted to deliver the composition as an aerosol to a lung of a person,wherein the composition comprises H₂O₂.
 2. The inhaler of claim 1wherein the composition further comprises thiocyanate.
 3. The inhaler ofclaim 1 wherein the composition further comprises a peroxidase.
 4. Theinhaler of claim 1 wherein the composition further comprises anantibiotic.
 5. The inhaler of claim 1 wherein the composition furthercomprises an anti-fungal or an antiviral.
 6. The inhaler of claim 1wherein the composition further comprises a sulfhydryl compound.
 7. Theinhaler of claim 1 wherein the composition further comprises apharmaceutically acceptable carrier.
 8. The inhaler of claim 2 whereinthe composition further comprises a pharmaceutically acceptable carrier.9. The inhaler of claim 3 wherein the composition further comprises apharmaceutically acceptable carrier.
 10. The inhaler of claim 4 whereinthe composition further comprises a pharmaceutically acceptable carrier.11. The inhaler of claim 5 wherein the composition further comprises apharmaceutically acceptable carrier.
 12. The inhaler of claim 6 whereinthe composition further comprises a pharmaceutically acceptable carrier.13. The inhaler of claim 1 further comprising a nozzle and an actuator.14. The inhaler of claim 2 further comprising a nozzle and an actuator.15. The inhaler of claim 3 further comprising a nozzle and an actuator.16. The inhaler of claim 4 further comprising a nozzle and an actuator.17. The inhaler of claim 5 further comprising a nozzle and an actuator.18. The inhaler of claim 5 further comprising a nozzle and an actuator.19. The inhaler of claim 7 further comprising a nozzle and an actuator.20. The inhaler of claim 8 further comprising a nozzle and an actuator.21. The inhaler of claim 9 further comprising a nozzle and an actuator.22. The inhaler of claim 10 further comprising a nozzle and an actuator.23. The inhaler of claim 11 further comprising a nozzle and an actuator.24. The inhaler of claim 12 further comprising a nozzle and an actuator.